Evidence that endogenous formaldehyde produces immunogenic and atherogenic adduct epitopes

Endogenous formaldehyde is abundantly present in our bodies, at around 100 µM under normal conditions. While such high steady state levels of formaldehyde may be derived by enzymatic reactions including oxidative demethylation/deamination and myeloperoxidation, it is unclear whether endogenous formaldehyde can initiate and/or promote diseases in humans. Here, we show that fluorescent malondialdehyde-formaldehyde (M2FA)-lysine adducts are immunogenic without adjuvants in mice. Natural antibody titers against M2FA are elevated in atherosclerosis-prone mice. Staining with an antibody against M2FA demonstrated that M2FA is present in plaque found on the aortic valve of ApoE mice. To mimic inflammation during atherogenesis, human myeloperoxidase was incubated with glycine, H O , malondialdehyde, and a lysine analog in PBS at a physiological temperature, which resulted in M2FA generation. These results strongly suggest that the 1,4-dihydropyridine-type of lysine adducts observed in atherosclerosis lesions are likely produced by endogenous formaldehyde and malondialdehyde with lysine. These highly fluorescent M2FA adducts may play important roles in human inflammatory and degenerative diseases

New insights into immunomodulation via overexpressing lipoic acid synthase as a therapeutic potential to reduce atherosclerosis

Atherosclerosis is a systemic chronic inflammatory disease. Many antioxidants including alpha-lipoic acid (LA), a product of lipoic acid synthase (Lias), have proven to be effective for treatment of this disease. However, the question remains whether LA regulates the immune response as a protective mechanism against atherosclerosis. We initially investigated whether enhanced endogenous antioxidant can retard the development of atherosclerosis via immunomodulation. To explore the impact of enhanced endogenous antioxidant on the retardation of atherosclerosis via immune regulation, our laboratory has recently created a double mutant mouse model, using apolipoprotein E-deficient (Apoe-/-) mice crossbred with mice overexpressing lipoic acid synthase gene (LiasH/H), designated as LiasH/HApoe-/- mice. Their littermates, Lias+/+Apoe-/- mice, served as a control. Distinct redox environments between the two strains of mice have been established and they can be used to facilitate identification of antioxidant targets in the immune response. At 6 months of age, LiasH/HApoe-/- mice had profoundly decreased atherosclerotic lesion size in the aortic sinus compared to their Lias+/+Apoe-/- littermates, accompanied by significantly enhanced numbers of regulatory T cells (Tregs) and anti-oxidized LDL autoantibody in the vascular system, and reduced T cell infiltrates in aortic walls. Our results represent a novel exploration into an environment with increased endogenous antioxidant and its ability to alleviate atherosclerosis, likely through regulation of the immune response. These outcomes shed light on a new therapeutic strategy using antioxidants to lessen atherosclerosis

A Gnotobiotic Mouse Model Demonstrates That Dietary Fiber Protects against Colorectal Tumorigenesis in a Microbiota- and Butyrate-Dependent Manner

It is controversial whether dietary fiber protects against colorectal cancer because of conflicting results from human epidemiologic studies. However, these studies and mouse models of colorectal cancer have not controlled the composition of gut microbiota, which ferment fiber into short-chain fatty acids such as butyrate. Butyrate is noteworthy because it has energetic and epigenetic functions in colonocytes and tumorsuppressive properties in colorectal-cancer cell lines. We utilized gnotobiotic mouse models colonized with wild-type or mutant strains of a butyrate-producing bacterium to demonstrate that fiber does have a potent tumor-suppressive effect but in a microbiota- and butyrate-dependent manner. Furthermore, due to the Warburg effect, butyrate was metabolized less in tumors where it accumulated and functioned as an HDAC inhibitor to stimulate histone acetylation and affect apoptosis and cell proliferation. To support the relevance of this mechanism in human cancer, we demonstrate that butyrate and histone-acetylation levels are elevated in colorectal adenocarcinomas compared to normal colonic tissues

Energy Balance Modulation Impacts Epigenetic Reprogramming, ERα and ERβ Expression, and Mammary Tumor Development in MMTV-neu Transgenic Mice

The association between obesity and breast cancer risk and prognosis is well established in ER-positive disease but less clear in HER2-positive disease. Here, we report preclinical evidence suggesting weight maintenance through calorie restriction may limit risk of HER2-positive breast cancer. In female MMTV-HER2/neu transgenic mice, we found that ERα and ERβ expression, mammary tumorigenesis and survival are energy balance-dependent in association with epigenetic reprogramming. Mice were randomized to receive a calorie restriction (CR), overweight (OW)-inducing, or diet-induced obesity (DIO) regimen (n = 27/group). Subsets of mice (n = 4/group/time point) were euthanized after 1, 3 and 5 months to characterize diet-dependent metabolic, transcriptional, and epigenetic perturbations. Remaining mice were followed up to 22 months. Relative to the OW and DIO regimens, CR decreased body weight, adiposity, and serum metabolic hormones as expected, and also elicited an increase in mammary ERα and ERβ expression. Increased DNA methylation accompanied this pattern, particularly at CpG dinucleotides located within binding or flanking regions for the transcriptional regulator CCCTC-binding factor (CTCF) of ESR1 and ESR2, consistent with sustained transcriptional activation of ERα and ERβ. Mammary expression of the DNA methylation enzyme DNMT1 was stable in CR mice but increased over time in OW and DIO mice, suggesting CR obviates epigenetic alterations concurrent with chronic excess energy intake. In the survival study, CR elicited a significant suppression in spontaneous mammary tumorigenesis. Overall, our findings suggest a mechanistic rationale to prevent or reverse excess body weight as a strategy to reduce HER2-positive breast cancer risk

A purified MAA-based ELISA is a useful tool for determining anti-MAA antibody titer with high sensitivity

Abstract Atherosclerosis is widely accepted to be a chronic inflammatory disease, and the immunological response to the accumulation of LDL is believed to play a critical role in the development of this disease. 1,4-Dihydropyridine-type MAA-adducted LDL has been implicated in atherosclerosis. Here, we have demonstrated that pure MAA-modified residues can be chemically conjugated to large proteins without by-product contamination. Using this pure antigen, we established a purified MAA-ELISA, with which a marked increase in anti-MAA antibody titer was determined at a very early stage of atherosclerosis in 3-month ApoE-/- mice fed with a normal diet. Our methods of Nε-MAA-L-lysine purification and purified antigen-based ELISA will be easily applicable for biomarker-based detection of early stage atherosclerosis in patients, as well as for the development of an adduct-specific Liquid Chromatography/Mass Spectrometry-based quantification of physiological and pathological levels of MAA

A purified MAA-based ELISA is a useful tool for determining anti-MAA antibody titer with high sensitivity

<div><p>Atherosclerosis is widely accepted to be a chronic inflammatory disease, and the immunological response to the accumulation of LDL is believed to play a critical role in the development of this disease. 1,4-Dihydropyridine-type MAA-adducted LDL has been implicated in atherosclerosis. Here, we have demonstrated that pure MAA-modified residues can be chemically conjugated to large proteins without by-product contamination. Using this pure antigen, we established a purified MAA-ELISA, with which a marked increase in anti-MAA antibody titer was determined at a very early stage of atherosclerosis in 3-month <i>ApoE</i>^<i>-/-</i> mice fed with a normal diet. Our methods of N^ε-MAA-L-lysine purification and purified antigen-based ELISA will be easily applicable for biomarker-based detection of early stage atherosclerosis in patients, as well as for the development of an adduct-specific Liquid Chromatography/Mass Spectrometry-based quantification of physiological and pathological levels of MAA.</p></div

Serum anti-MAA IgG and IgM antibody levels in <i>wild-type</i> and <i>ApoE</i>^-/- mice.

<p><b>(A)</b> Serum anti-MAA IgG and IgM antibody levels in wild-type and <i>ApoE</i>^-/- mice. The anti-MAA IgG and IgM antibody levels showed significant differences between <i>wild-type</i> and <i>ApoE</i>^<i>-/-</i> mice with the pMAA-6ACA-BSA and crMAA-BSA ELISAs for all except the IgM levels with crMAA-BSA ELISA (*: p<0.05; **: p<0.01). In addition, the differences between pMAA-6ACA-BSA and crMAA-BSA ELISAs in the <i>wild type</i> and <i>ApoE</i>^<i>-/-</i> mice for anti-MAA IgG and IgM antibody levels were significant for all except the IgM levels in <i>ApoE</i>^<i>-/-</i> mice. <b>(B)</b> The anti-MAA IgG and IgM antibody titers in <i>ApoE</i>^-/- mice were normalized to those of <i>wild-type</i> mice. The individual titer values of <i>ApoE</i>^-/- animals were divided by the average titer value of the <i>wild-type</i> animals. Of note, the differences between the antibody titers in the <i>wild-type</i> and <i>ApoE</i>^-/- mice were much greater with the pMAA-6ACA-BSA ELISA than with the crMAA-BSA ELISA. Error bars represent SD. Due to the redundancy of the statistical analyses, we did not include any asterisks for statistical significance in Fig 5B.</p

The full scan mass spectrums of MAA-lysine and MAA-6ACA.

<p>(<b>A</b>) The full scan mass spectrum shows the protonated molecular ion for MAA-lysine at <i>m/z</i> 281.1498. Other peaks are minor background ions and reference ions <i>m/z</i> 121.0509 and <i>m/z</i> 922.0098. (<b>B</b>) The full scan mass spectrum shows the protonated molecular ion of MAA-6ACA at <i>m/z</i> 266.1391. Other peaks are minor background ions and reference ions <i>m/z</i> 121.0509 and <i>m/z</i> 922.0098.</p

The immunogenicity of pMAA-lysine-BSA in the absence of adjuvant.

<p>C57BL/6 mice were injected i.p. with pMAA-lysine-BSA or BSA in the absence of adjuvant. The antibody titers of IgG (<b>A</b>) and IgM (<b>B</b>) against pMAA-lysine were detected using pMAA-6ACA-KLH-coated plates. The anti-MAA antibody titers were clearly increased in pMAA-lysine-BSA-immunized mice compared to the controls (BSA-treated mice). Error bars represent SD.</p